US5256556A - Process for obtaining invertase from yeast - Google Patents

Process for obtaining invertase from yeast Download PDF

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Publication number
US5256556A
US5256556A US07/573,222 US57322290A US5256556A US 5256556 A US5256556 A US 5256556A US 57322290 A US57322290 A US 57322290A US 5256556 A US5256556 A US 5256556A
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Prior art keywords
invertase
yeast
disrupted
cell suspension
heat treatment
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Expired - Fee Related
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US07/573,222
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English (en)
Inventor
Helmut Hustedt
Kay Buntemeyer
Karl-Heinz Kroner
Bernhard Borner
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Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
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Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification

Definitions

  • the invention relates to methods of preparing invertase from yeast.
  • Invertase is contained within the cells of various yeasts. In order to obtain the enzyme, which is employed on the industrial scale in the foodstuffs industry, it is necessary to disrupt the yeast cells. This is followed by removal of the cell detritus and undesired proteins.
  • Invertase is distinguished by an above-average resistance to heat and low pH values. Based on this, a process for obtaining invertase has already been proposed, in which a heat treatment is carried out at a pH of 5.0, but this yields a purification factor of only 2. It is therefore not surprising that the method of obtaining invertase in practice does not make use of this process which was proposed as long ago as 1967; N.P. Neumann and J.O. Lampen, Biochemistry 6 (1967), 468-475 . On the contrary, the classical working up of invertase provides for two acetone-precipitation steps; cf., for example, Ullmanns Enzyklopadie der Ü Chemie; H. Vetter et al. "Enzyme”, in: Ullmanns Enzyklopadie der ischen Chemie, Vol. 10, pp. 475-561 Verlag Chemie, Weinheim, 1975.
  • a process for obtaining invertase from yeast is now proposed according to the invention, in which the yeast cells are disrupted, the disrupted cell suspension is subjected in a strongly acid medium to a heat treatment, and the denatured undesired proteins are removed with the cell detritus, preferably by centrifugation, and then the invertase is isolated where appropriate, this process being characterized in that
  • the disrupted cell suspension is subjected at a pH of less than 4.5
  • the disrupted cell suspension is subjected to an acid treatment at a pH of less than or equal to 4.0 without heat treatment.
  • Preferably used in the heat treatment is a temperature in the range from 45° to 50° C. at a pH in the range from 3.0 to 4.2, with a temperature of 48° to 50° C. and the pH range 3.8 to 4.2 being particularly preferred.
  • Suitable yeasts are brewer's yeast, baker's yeast or yeasts derived therefrom, or microorganisms which contain the genetic information for the production of brewer's yeast invertase or baker's yeast invertase.
  • FIG. 1 is a graphical representation of the enzyme activity and protein content of preparations subjected to acid and heat treatment.
  • FIG. 3 is a graphical representation of yield and purity of preparations subjected to heat-treatment (various pH).
  • FIG. 4 is a graphical representation of enzyme stability in acid medium.
  • FIG. 6 is a graphical representation of enzyme activity, purity and protein content after preparation with heat treatment.
  • FIG. 7 is a graphical representation of enzyme activity, purity and protein content of preparations made in accordance with the invention.
  • the raw material used was fresh, commercially obtainable baker's yeast (DHW, Northeim) which was supplied in 25 kg bags.
  • the material contained, distributed inhomogeneously, small amounts of kieselguhr which derived from the cell harvest.
  • the yeast was suspended in a 1/10 molar dipotassium hydrogen phosphate buffer.
  • the suspension had a pH of approximately 7.25.
  • Disruption was then carried out in a high-pressure homogenizer (Gaulin M 3) until about 75 g of protein had been released per kg moist mass of cells.
  • the disruption was carried out in each case by three passages through the homogenizer at 550 bar.
  • the homogenisate was cooled to about 15° C. in a continuous heat exchanger immediately after leaving the homogenizer.
  • the disrupted cell suspension was adjusted to a pH of 4 in each case with phosphoric acid or with acetic acid by titration in a stirred container.
  • the titrated suspension was stirred for about half an hour at a stirrer power of about 4 kW/m 3 .
  • the heat treatment was carried out in a continuous thermal denaturation system, specifically in a static tubular mixer/heat exchanger which ensured rapid and uniform heating with few temperature peaks.
  • the residence time distribution resulted in a mean treatment time of 10 minutes with a minimum time of 9 minutes and a maximum time of 11 minutes for 70% of the product.
  • the temperature of the product at the exit from the heater was about 51° C. (0.5° C. standard deviation).
  • the holding section the product cooled to an exit temperature of 44° C., which was reached after 10 minutes. Cooling to about 15° C. then took place in a second heat exchanger.
  • a 0.05 molar acetate buffer solution was titrated with 2.5 normal potassium hydorxide solution to a pH of 4. This buffer was used to dilute the heat-treated suspension to a content of 10% moist mass of cells.
  • the diluted suspension was worked up using a disk separator (1500 m 2 equivalent clarification area; Westphalia SA-1). Sediment was removed batchwise from the separator, with 17.6% by volume of the suspension being removed as sediment. The enzyme loss in this case was at a similar level. The throughput was 36 1/h.
  • the separator residue was subjected to an ultrafiltration for concentration.
  • An ultrafiltration unit of polysulfone material with an area of 0.8 m 2 was used for this. Since invertase has a molecular weight of 270,000 dalton, adequate retention is achieved with an ultrafiltration membrane with a pore size of 1000,000 dalton. With a pressure difference of 1 bar across the membrane and a retentate flow rate of 370 1/h, the average filtrate flow rate achieved was from 60 1/h m 2 to 75 1/h m 2 .
  • the permeate had an invertase activity of about 3 U/ml, which was equivalent to a 4% loss of product. It was possible with the chosen design of experiment to concentrate the product by a factor of 14.75, with about 50 1 of separator supernatant being worked up.
  • FIGS. 1 to 7 and Table 2 which follow further demonstrate and explain the invention.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US07/573,222 1989-05-12 1990-05-10 Process for obtaining invertase from yeast Expired - Fee Related US5256556A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3915616A DE3915616C1 (de) 1989-05-12 1989-05-12
DE3915616 1989-05-12

Publications (1)

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US5256556A true US5256556A (en) 1993-10-26

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US (1) US5256556A (de)
EP (1) EP0447497A1 (de)
DE (1) DE3915616C1 (de)
WO (1) WO1990013636A1 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120284A1 (en) * 2014-02-07 2015-08-13 Qol Medical Llc Ultrapure hypoallergenic solutions of sacrosidase

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1689607A (en) * 1925-11-06 1928-10-30 John J Naugle Invertase preparation and method of preparing and utilizing the same
US1855591A (en) * 1926-02-03 1932-04-26 Wallerstein Co Inc Invertase preparation and method of making the same
US1855592A (en) * 1926-02-03 1932-04-26 Wallerstein Co Inc Invertase preparation and method of making the same
US1919675A (en) * 1929-08-30 1933-07-25 Wallerstein Co Inc Invertase preparation and method of making the same
US1919676A (en) * 1931-09-30 1933-07-25 Wallerstein Co Inc Method of making invertase preparations
US1990505A (en) * 1929-08-30 1935-02-12 Wallerstein Co Inc Method of making an invertase preparation
US2164936A (en) * 1937-03-12 1939-07-04 Standard Brands Inc Process for the separation of invertase
US3427223A (en) * 1964-06-10 1969-02-11 Exxon Research Engineering Co Coagulating microbial cells to enhance their separation
US3887434A (en) * 1972-06-09 1975-06-03 Bayer Ag Microbiological production of invertase
US4278764A (en) * 1978-10-27 1981-07-14 Euteco Impianti S.P.A. Process for preparing citric acid by fermentation of carbohydrates
GB2198731A (en) * 1986-11-20 1988-06-22 Halle Ingenieurtech Preparation of invertase from yeast
US4925796A (en) * 1986-03-07 1990-05-15 Massachusetts Institute Of Technology Method for enhancing glycoprotein stability

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE640455C (de) * 1935-03-31 1937-01-05 I G Farbenindustrie Akt Ges Verfahren zur Herstellung gereinigter Invertaseloesungen
AU566747B2 (en) * 1984-01-10 1987-10-29 Mitsui Chemicals, Inc. Method of separating l-tryptophan

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1689607A (en) * 1925-11-06 1928-10-30 John J Naugle Invertase preparation and method of preparing and utilizing the same
US1855591A (en) * 1926-02-03 1932-04-26 Wallerstein Co Inc Invertase preparation and method of making the same
US1855592A (en) * 1926-02-03 1932-04-26 Wallerstein Co Inc Invertase preparation and method of making the same
US1919675A (en) * 1929-08-30 1933-07-25 Wallerstein Co Inc Invertase preparation and method of making the same
US1990505A (en) * 1929-08-30 1935-02-12 Wallerstein Co Inc Method of making an invertase preparation
US1919676A (en) * 1931-09-30 1933-07-25 Wallerstein Co Inc Method of making invertase preparations
US2164936A (en) * 1937-03-12 1939-07-04 Standard Brands Inc Process for the separation of invertase
US3427223A (en) * 1964-06-10 1969-02-11 Exxon Research Engineering Co Coagulating microbial cells to enhance their separation
US3887434A (en) * 1972-06-09 1975-06-03 Bayer Ag Microbiological production of invertase
US4278764A (en) * 1978-10-27 1981-07-14 Euteco Impianti S.P.A. Process for preparing citric acid by fermentation of carbohydrates
US4925796A (en) * 1986-03-07 1990-05-15 Massachusetts Institute Of Technology Method for enhancing glycoprotein stability
GB2198731A (en) * 1986-11-20 1988-06-22 Halle Ingenieurtech Preparation of invertase from yeast

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Cheremisinoff et al., Biotechnology: Applications and Research , 1985, pp. 534 546. *
Cheremisinoff et al., Biotechnology: Applications and Research, 1985, pp. 534-546.

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120284A1 (en) * 2014-02-07 2015-08-13 Qol Medical Llc Ultrapure hypoallergenic solutions of sacrosidase
US9255261B2 (en) 2014-02-07 2016-02-09 Qol Medical Llc Ultrapure hypoallergenic solutions of sacrosidase
US9469847B2 (en) 2014-02-07 2016-10-18 Qol Medical Llc Ultrapure hypoallergenic solutions of sacrosidase
US9849161B2 (en) 2014-02-07 2017-12-26 Qol Medical Llc Ultrapure hypoallergenic solutions of sacrosidase
EP3417873A1 (de) * 2014-02-07 2018-12-26 QOL Medical LLC Ultrareine hypoallergene lösungen von sacrosidase
US10588947B2 (en) 2014-02-07 2020-03-17 Qol Medical Llc Ultrapure hypoallergenic solutions of sacrosidase

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Publication number Publication date
DE3915616C1 (de) 1990-06-21
EP0447497A1 (de) 1991-09-25
WO1990013636A1 (de) 1990-11-15

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